| Objective: To investigate morphology and p53 protein expression during the malignant transformation of immortalized human bronchial epithelial cell (BEAS-2B) induced by tobacco-specific nitrosamine (NNK), and explore the significance of early-warning and mass screening of lung cancer in high-risk smoking population. Methods: Immortalized human bronchial epithelial cells (BEAS-2B)were induced to malignant transformation cells (BEAS-2BNNK) by NNK, and p53 protein expression was detected in the different passages of BEAS-2BNNK and BEAS-2B cells by Sp immunocytochemistry. The cell morphology and parameters (perimeter, maximum diameter, minimum diameter, ratio of karyoplasm and form factor) were observed under microscope and electron microscope in each group. Results: 1.The model of malignant transformation of BEAS-2B cells induced by NNK was created. ①The serum resistance was significantly increased in the 5th passage BEAS-2BNNK cells. ② The anchorage independent growth(soft agar colony formation)was appeared in the 15th passage. ③ The 25th passage BEAS-2BNNK cells formed tumors in nude mice, and tumors were squamous cell carcinoma(I-II grade) confirmed by histopathological examination. 2. Morphology change ①Microstructure: The microstructure of BEAS-2B cells was not significantly changed in different passages. Compared with the corresponding BEAS-2B cells, the shape of cell clone, the way of cell growth, the shape and quantity of cell and nuclear were not significantly changed in the 1st, 2nd, 5th passage BEAS-2BNNK cells. The shapes of cells and its nuclears of the 10th, 15th, 20th, 25th passage BEAS-2BNNK cells became round gradually, and the way of cell growth became from radio-form into membraniform and more than one nucleolus appeared. ②Ultrastructure change: The ultrastructure of BEAS-2B cells was not significantly changed in different passages. Compared with the corresponding BEAS-2B cells, the shapes of cells and nuclears, the shapes and quantity of cell organs were not significantly changed in the 1st, 2nd, 5th passage BEAS-2BNNK cells. The 10th, 15th passage BEAS-2BNNK cells, which had swelled cell and cell organs and abnormal nuclear and enlarged the nucleoli gradually and increased the quantity of the cell organs and activated their function, showed the characteristic of transformation cells. The 20th, 25th passage BEAS- 2BNNKcells, which had obvious aberration of the cell and nucleoli and had cataclasm of nucleoli and decreased cell organs, showed the characteristic of tumor cells. ③Morphological parameters: Morphological parameters of different passages of BEAS-2B cells were no difference in statistics. And the morphological parameters were also no difference between BEAS-2B cells and BEAS-2BNNKcells in 1st, 2nd, 5th passage(p>0.05). Compared with BEAS-2B cells, the morphological parameters of BEAS-2BNNK cells became large gradually in 10th, 15th passage (p<0.05), especially in 20th, 25th passage (p<0.01). 3. p53 protein expressions: p53 protein expression of BEAS-2B cells was negative, but positive in the different passage BEAS-2BNNKcells. With the passage of BEAS-2BNNK cells increased gradually, p53 protein expression rised more and more obviously(p<0.001). Conclusion: 1. The model of malignant transformation of BEAS-2B cells induced by NNK(500μɡ/ml) could be created successfully. 2. Biology characteristic of transformation cells in middle and late passages could be accurately, objectively and quantitatively judged by microstructure, ultrastructure and morphological parameters, but was difficult to judge in earlier passages. 3. There were obvious p53 protein expressions in earlier passages. With the passage of BEAS-2BNNKcells increased gradually, p53 protein expression rised more and more obviously. 4. During the malignant transformation, the change of morphological characteristic of BEAS- 2BNNKcells was later than that of p53 protein expression. 5. The characteristic of p53 being expressed before BEAS-2BNNK cells changed into cancer cells wa...
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